Abstract

The Sfpi1 gene encodes PU.1, a critical transcription factor in multiple hematopoietic lineages. PU.1 expression is upregulated as hematopoietic stem cells become granulocyte-macrophage progenitors. In contrast, Sfpi1 must be silenced after progenitors undergo T-lineage specification. If unrestrained in early T-lineage cells, PU.1 can both block developmental progress and induce diversion to a myeloid fate. When PU.1 expression is not sufficiently increased or maintained in myeloid lineage cells, myeloid hyperproliferation and cancer can result. In mouse DN thymocytes, PU.1 mRNA begins at high levels in early T-cell progenitors, but drops about fivefold as cells enter the T-cell program (DN2) and then falls tenfold further as the cells reach T-lineage commitment (DN3). This implies operation of a stage-specific repression mechanism correlated with commitment. Only one major cis-regulatory element has previously been described for Sfpi1, which is a compound conserved region around -14 kb that is thought to mediate activation as well as some repression. However, it cannot account for all PU.1 regulation in early T-lineage cells nor in myeloid cells. In particular, that -14 kb element can show strong enhancer activity in an immature T-cell line in which the endogenous Sfpi1 gene is profoundly repressed. Additionally, absence of the -14 kb element does not abolish PU.1 expression in myeloid lineages. We now present evidence for another complex of conserved noncoding elements that appear to mediate several cell-type-specific regulatory features, including cell-type-specific repression in early T-cells. We describe fine mapping of a T-cell specific bipartite silencer and show that the T lineage specific repressive activity requires Runx1. We also describe additional regulatory complexes that may contribute to lineage specific regulation of PU.1 in early hematopoietic progenitors, including a myeloid specific enhancer. We provide evidence of lineage restricted occupancy of these additional regulatory elements and show that the novel enhancer elements are additional sites of PU.1 auto regulation.